Mitogen-activated protein kinase (MAPK) signaling pathways participate in the regulation of many biologically and medically important processes, including normal and pathological aspects of cell growth, division, differentiation, and death. Their ubiquity and versatility raise the issue of how they achieve specific coupling of signal to cellular response. How do the kinases in the cascade distinguish their correct substrates from the vast excess of incorrect substrates? Furthermore, how do different signals elicit distinct responses when they are transmitted by the same components? This competitive renewal proposal presents experiments that address the specificity of recognition between MAP kinases and their substrates and regulators, and that investigate the mechanisms that restrict inappropriate signal crossover (or 'leaking') between distinct pathways sharing components. We continue to investigate both yeast and mammalian MAPK pathways, in order to innovatively exploit the synergistic cross-fertilization of insights gained from these two experimental systems. Specific Aims are (1) to delineate the selectivity of MAPK-docking sites ('D-sites') in MAPK kinases (MKKs, or MEKs) for their cognate vs. non-cognate MAPKs and to develop a model to explain these relationships; (2) to assess MEK D-site function and specificity in vivo; (3) to identify new substrates and regulators of the JNK MAP kinase by predicting JNK-docking sites; (4) to determine the molecular mechanism of Fus3MAPK-dependent feedback control of specificity in yeast; (5) to investigate how the yeast Ste5 scaffold protein contributes to signaling specificity; (6) to ask if the MAPKs Kss1 and Fus3 have distinct target site preferences. Chronic activation, loss of sensitivity to feedback controls and loss of specificity appear to be important aspects of the MAPK dysregulation seen in many cancers, and this research will increase our understanding of these issues. More generally, this research could lead to a better understanding of how MAP kinases (and other kinases) find their targets and improve our ability to predict substrates and regulators and may ultimately suggest novel approaches to therapy based on the modulation of protein kinase interactions.